CN101597633B - New method for producing recombinant human hematopoietin by cell suspension culture - Google Patents
New method for producing recombinant human hematopoietin by cell suspension culture Download PDFInfo
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- CN101597633B CN101597633B CN2008100646526A CN200810064652A CN101597633B CN 101597633 B CN101597633 B CN 101597633B CN 2008100646526 A CN2008100646526 A CN 2008100646526A CN 200810064652 A CN200810064652 A CN 200810064652A CN 101597633 B CN101597633 B CN 101597633B
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Abstract
The invention relates to cell culture in a producing process of recombinant human hematopoietin, in particular to a method for domesticating cell strains generated by recombinant human hematopoietin to adapt to serum-free culture. A disposable pocket type torrent reactor is used for the large-scale suspension culture of cells so as to increase the growth density of the cells, and thereby the recombinant human hematopoietin is effectively produced. The method can increase the batch production quantity, shorten the production period and reduce cost and pollution.
Description
Affiliated technical field
The present invention relates to the cell culture processes in the recombinant human erythropoietin production process; Especially cell strain adaptation serum-free culture is produced in domestication, carries out suspension culture with disposable pocket type torrent reactor drum pair cell, improves the cell stand density; Shorten the production cycle; Reduce production costs, reduce and pollute, thus the High-efficient Production recombinant human erythropoietin.
Background technology
Hempoietine is a kind of glycoprotein hormones, the about 34kD of molecular weight.The erythropoietin that exists in the blood plasma is made up of 165 amino acid, and degree of glycosylation is very high, and the glycosyl composition mainly is a sialyl.Different according to carbohydrate content, naturally occurring erythropoietin is divided into two types, and the α type contains 34% glucide, and the β type contains 26% glucide.Two types all identical on biological characteristics, antigenicity and clinical application effect.Human erythropoietin gene is positioned at long 22 districts of karyomit(e) No. 7, and its cDNA was successfully cloned in 1985.Hempoietine is mainly synthetic by the uriniferous tubules endotheliocyte, also can be produced by liver cell and scavenger cell.Erythropoietin is that a kind of hematopoiesis of strong effect generates the factor, when 0.05～1U/ml, is dose-dependent effect.The erythropoietin activity is single, only acts on marrow macronucleus precursor cell, can stimulate red progenitor cell and basophilic normoblast to be formed into ripe red corpuscle colony; Adjusting to the red corpuscle hematopoiesis needs the synergy of other cytokines (like IL-3, GM-CSF and IL-1 etc.) to accomplish.The generation of erythropoietin receives the adjusting of Q volume of blood and oxygen partial pressure in the body, lose blood or the stimulation of hypoxemia under, the erythropoietin level rises rapidly.At some tumour patient, can also show erythropoietin and increase unusually; The anaemia patient of marrow hemopoiesis poor response, its erythropoietin also raises.Recombinant human erythropoietin is that by the albumen that BA is arranged of Chinese hamster ovary celI fermentation expression, it has the biological function similar with the human body endogenous erythropoietin through the recombination human erythropoietin gene of genetically engineered human cloning.
At present, in the substratum application facet, recombinant human erythropoietin is produced cell strain and mainly in the substratum that contains 8～10% Ox blood serums, is carried out amplification cultivation, and cell amplification is changed to serum free medium after a certain amount of and cultivates.Wherein, serum has a lot of insufficient places, such as:
1. the composition of serum has more than the hundreds of kind, and composition, content and mechanism of action thereof are still unclear accurately to it at present, especially some of them peptide growth factor, hormone and lipid etc. are not fully realized as yet, and work brings many difficulties to purifying for this.
2. serum all is to produce in batches, and is widely different between each batch, and the serum preservation period only one year, therefore, guarantee that the similarity of every batch of serum is very difficult, thereby the stdn of production and continuity are restricted.
3. animal individual is different, the serum place of production, lot number difference, and every quality of lot difference is very big, and its composition can not be consistent.
4. possibly bring mycoplasma, virus in drawing materials, pair cell produces potential impact, possibly cause producing failure or production unreliability as a result.
5. in the scale operation, serum source more and more difficult costs an arm and a leg, and is one of the major portion that constitutes the production cost of animal cell culture.
The serum free medium quality is more stable relatively, can improve the repeatability of cell cultures and experimental result.Avoided the serum source contact scar.Can improve the expression level of product and make cellular product be easy to purifying, have a lot of serum free mediums available at present on the market.
Cell must be attached on the surface of matrix and could grow when cell is having adherent growth in the blood serum medium, once cell grows up to individual layer, because intercellular contact inhibition, cell is no longer bred.Cell density when cell attachment is grown like this depends on the size that attaches substrate surface area, and every square centimeter of surface-area can hold 1～5 * 10
5Individual cell, therefore, in square vase and rolling bottle cultivation, cell density generally can only be 1～5 * 10
6Between the cells/ml.In the bio-reactor that has adopted microcarrier or chip carrier, cell density can reach 1～2 * 10
7Cells/ml.
Yet in serum free medium, cell can carry out suspension culture through domestication, and cell density depends primarily on the nutrition degree of substratum, and therefore, theoretically, as long as enough nutrition is arranged, cell density can be infinitely great.At present, for different serum free mediums, cell density can reach 0.5～1 * 10
8Between the cells/ml.
At present, what recombinant human erythropoietin production was mainly used is NBS bio-reactor (or rolling bottle), in tank body, adds microcarrier and carries out adherent perfusion cultivation; Culture cycle is about 2 months, and the recombinant human erythropoietin expression amount is 10mg/L, and this training method is because the tank body volume is little; The microcarrier amount is limited, and cell density is lower, and the recombinant human erythropoietin expression amount is low; Culture cycle is longer, and production cost is high.
Summary of the invention
Disposable pocket type torrent reactor drum described in the present invention is meant patent " a kind of cell suspension culture tank " (application number: 200710130135.X) said related prods of Harbin Anpu Science & Technology Development Co., Ltd's application.
It is high to the purpose of this invention is to provide a kind of cell density, amplifies easily, with short production cycle, the recombinant human erythropoietin working method that expression amount is high.The present invention makes it suspension growth in serum free medium, thereby has changed the mode of cell cultures through domestication recombinant human recombinant human erythropoietin expression cell line, reaches high-density culturing cell, the scale operation recombinant human erythropoietin.
The invention provides cell suspension culture and produce the method for recombinant human erythropoietin, this method comprises the steps:
A) the recombinant human erythropoietin expression cell line is in the less serum-free culture in the bottle of shaking;
B) when cell grows into certain density, be inoculated into serum-free culture in the bigger sack that shakes bottle or torrent reactor drum;
C) make cell proliferation, express;
D) purifying results recombinant human erythropoietin.
This working method is not used serum, and the substratum quality is stable, can improve product production.Because with disposable pocket type torrent reactor drum suspension culture, the cell growth does not receive the long-pending restriction of wall-attached surface, cell density is higher than adherent growth (5～10 times) far away; The recombinant human erythropoietin expression amount is high; Amplify easily, reduced production cost, avoided the pollution of serum.
Embodiment:
Under many circumstances, cell is preserved in serum is arranged, and needs pair cell to tame; Make it adapt to suspension growth in serum-free; Domestication is the very conventional technology in this area, and concrete steps are following: with substratum (DMEM substratum) passage cell that contains serum (foetal calf serum) several times, get the cell that is in logarithmic phase then and process suspension earlier; Receive aseptic shaking in the bottle, suitable inoculum density is 1～5 * 10
5Cells/ml, the speed of shaking table are 100-400rmp, and suitable speed is 200-300rmp, when cell density reaches 1～5 * 10
6During cells/ml, carry out passage.The best density that goes down to posterity is 2～3 * 10
6, went down to posterity by 1: 3, when cell speed of growth in serum-free, with contain in the serum speed of growth near the time, just realized the domestication of cell.The present invention is to equipment, and reagent does not have particular requirement.
Further specify the present invention through embodiment below, the present invention is become restriction inadequately.
Embodiment 1
Recombinant human erythropoietin cell strain of recovery adds 10% foetal calf serum with the DMEM substratum and goes down to posterity twice from cell bank, when cell is in logarithmic phase, uses tryptic digestion; Increase serum stops digestion, and 1000rmp is 5 minutes, centrifugal; Remove supernatant, add serum free medium (Harbin Anpu Science & Technology Development Co., Ltd provides), process suspension to cell; Be inoculated into aseptic shaking in the bottle, every flask culture matrix is long-pending to be 50 milliliters, and inoculating cell density is 3 * 10
5Cells/ml, shaking table speed are 200rmp, when cell density reaches 2～3 * 10
6Cells/ml went down to posterity by 1: 3, went down to posterity 5～6 times the time, and when cell speed of growth in serum-free, the speed of growth is approaching with containing in the serum, freeze-stored cell.Set up the seed cell storehouse.
Embodiment 2
Recombinant human erythropoietin cell strain of recovery adds 10% foetal calf serum with the DMEM substratum and goes down to posterity twice from cell bank, when cell is in logarithmic phase, uses tryptic digestion; Increase serum stops digestion, and 1000rmp is 5 minutes, centrifugal; Remove supernatant, add serum free medium (CHO of GIBCO company serum-free suspension culture base), process suspension to cell; Be inoculated into aseptic shaking in the bottle, every flask culture matrix is long-pending to be 50 milliliters, and inoculating cell density is 3 * 10
5Cells/ml, shaking table speed are 200rmp, when cell density reaches 2～3 * 10
6Cells/ml went down to posterity by 1: 3, went down to posterity 5～6 times the time, and when cell speed of growth in serum-free, the speed of growth is approaching with containing in the serum, freeze-stored cell.Set up the seed cell storehouse.
Embodiment 3
Recombinant human erythropoietin cell strain of recovery adds 10% foetal calf serum with the DMEM substratum and goes down to posterity twice from cell bank, when cell is in logarithmic phase, uses tryptic digestion; Increase serum stops digestion, and 1000rmp is 5 minutes, centrifugal; Remove supernatant, add serum free medium (the serum-free suspension culture base of SAFC Bioscience company), process suspension to cell; Be inoculated into aseptic shaking in the bottle, every flask culture matrix is long-pending to be 50 milliliters, and inoculating cell density is 3 * 10
5Cells/ml, shaking table speed are 200rmp, when cell density reaches 2～3 * 10
6Cells/ml went down to posterity by 1: 3, went down to posterity 5～6 times the time, and when cell speed of growth in serum-free, the speed of growth is approaching with containing in the serum, freeze-stored cell.Set up the seed cell storehouse.
Embodiment 4
Used substratum is that Harbin Anpu Science & Technology Development Co., Ltd provides in the present embodiment; (20L) carries out the coffee radiation sterilization to sack; Adding substratum is that Harbin Anpu Science & Technology Development Co., Ltd provides serum free medium 10L; And the seed cell of gained among the inoculation embodiment 1, inoculum density is 4 * 10
5Cells/ml, at 37 degree, rotating speed is to cultivate under the 200rmp, and stream adds concentrated substratum (Harbin Anpu Science & Technology Development Co., Ltd provides serum free medium), and survey sugar every day, guarantees sugared content at 0.5～1g/L, sufficient to protect cytotrophy.
Cultivate after 5～8 days, cell density can reach 3～8 * 10
7Cells/ml continues to cultivate 2～3 days results supernatants, carries out purifying, and the recombinant human erythropoietin expression amount reaches 25000IU/ml, and adherent culture is 5000～10000IU/ml, and other indexs all have raising.
Embodiment 5
Used substratum is that Harbin Anpu Science & Technology Development Co., Ltd provides in the present embodiment; (40L) carries out the coffee radiation sterilization to sack; Adding substratum is that Harbin Anpu Science & Technology Development Co., Ltd provides serum free medium 20L; And the seed cell of gained among the inoculation embodiment 1, inoculum density is 4 * 10
5Cells/ml, at 37 degree, rotating speed is to cultivate under the 200rmp, and stream adds concentrated substratum (Harbin Anpu Science & Technology Development Co., Ltd provides serum free medium), and survey sugar every day, guarantees sugared content at 0.5～1g/L, sufficient to protect cytotrophy.Cultivate after 5～8 days, cell density can reach 3～8 * 10
7Cells/ml continues to cultivate 2～3 days results supernatants, carries out purifying, and the recombinant human erythropoietin expression amount reaches 25000IU/ml, and adherent culture is 5000～10000IU/ml, and other indexs all have raising.
Claims (1)
1. the method for a cell suspension culture recombinant human erythropoietin is characterized in that, this method comprises the steps:
(1) recombinant human erythropoietin cell strain of recovery from cell bank adds 10% foetal calf serum with the DMEM substratum and goes down to posterity twice, when cell is in logarithmic phase, uses tryptic digestion; Increase serum stops digestion, 1000rpm, centrifugal 5 minutes; Remove supernatant, add serum free medium, process suspension to cell; Be inoculated into aseptic shaking in the bottle, every flask culture matrix is long-pending to be 50 milliliters, and inoculating cell density is 3 * 10
5Individual cells/ml, shaking table speed are 200rmp, when cell density reaches 2～3 * 10
6Cells/ml went down to posterity by 1: 3, go down to posterity 5～6 times the time, the speed of in serum free medium, growing when cell, with the speed of in containing blood serum medium, growing near the time, freeze-stored cell, as seed cell,
(2) sack to torrent formula bio-reactor carries out the coffee radiation sterilization, adds serum free medium 10L, and the inoculation seed cell, and inoculum density is 4 * 10
5Individual cells/ml, at 37 degree, rotating speed is to cultivate under the 200rmp, and stream adds spissated serum free medium, and survey sugar every day, guarantees sugared content at 0.5-1g/L, cultivates after 5-8 days, and cell density can reach 3-8 * 10
7Cells/ml continues to cultivate 2-3 days results supernatants, purifying results recombinant human erythropoietin.
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| CA2800284C (en) * | 2010-05-25 | 2020-02-11 | Cook Medical Technologies Llc | Methods, substrates, and systems useful for cell seeding of medical grafts |
| CN103103237B (en) * | 2011-11-09 | 2014-11-12 | 哈药集团技术中心 | Method for cell perfusion culture to produce recombinant protein by microcarrier technology |
| CN105462909B (en) * | 2015-12-31 | 2019-09-27 | 哈药集团技术中心 | A kind of cultural method of the Chinese hamster ovary celI of high efficient expression Human Fallicle-Stimulating Hormone |
| CN114317431A (en) * | 2022-01-03 | 2022-04-12 | 中国人民解放军军事科学院军事医学研究院 | Method for promoting differentiation of human hematopoietic stem/progenitor cells to erythroid line |
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| CN1898379A (en) * | 2003-12-23 | 2007-01-17 | 先灵公司 | Methods for producing cell lines stable in serum-free medium suspension culture |
| CN1570090A (en) * | 2004-05-11 | 2005-01-26 | 戴育成 | Cell suspension culture and anchorage culture serum free medium and its preparation method |
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